Scorpions are distributed throughout the tropical and subtropical belts of the world in habitats ranging from dry deserts to the mountains. Only a fraction of the existing species have venom potent enough to endanger humans and almost all of these are found in the family Buthidae. Those considered most dangerous are found in the Middle East, Asia, South America, and Africa. Parabuthus transvaalicus is a large (up to 150 mm) South African scorpion species from the family Buthidae and considered to be medically important (Bergman (1997) Toxicon 35:759–771). The victims of a sting by P. transvaalicus can suffer from neurotoxic effects, prolonged pain, which lasts from one day in minor cases and up to a week in severe cases, and even death. Symptoms include abnormal reflexes, bladder symptoms, dysphagia, sweating and hypersalivation (Bergman, N. J. (1997), supra).
Venom of P. transvaalicus is a ‘simple’ venom compared to other scorpion venoms because it contains less than 100 major peptides. The venom may be characterized by defining the individual components of the system (identification of peptide toxins), analysis of the structure of the components (primary, secondary and tertiary structure determination), analysis of the function of each component (determination of the mode of action), analysis of the relationships between these components (synergism) and the target sites or the environment (binding sites and kinetics).
Although poisonous scorpions are sprinkled across several genera taxonomically, the action of the venom is similar. Scorpion venoms are a rich source of neurotoxic peptides with diverse modes of action. Within the complex mixture of venoms, peptides have been found-to possess the majority of the biological effect towards the sting victims; however, these peptides are usually low in abundance (Nakagawa (1997) Eur. J. Biochem. 246:496–501). Stings manifest themselves mostly in the peripheral nervous system, resulting in symptoms such as intense pain at the sting site, altered heart activity, and parasthesia. Stings to children, the elderly, and unhealthy individuals are much more dangerous and more often lethal. Where antivenom is available, it is very effective in counteracting the effects of the sting, and when administered, victims are typically asymptomatic within 90 minutes.
Current methods for antivenom production involve the direct injection into horses of crude venom or antibodies produced from a mixture of a number of species' venom. However, there are risks associated with the injection of antibodies from another animal, or passive immunization. The recipient can mount a strong immunologic response to the isotypic determinants of the foreign antibody. This anti-isotype response can have serious complications because some recipients will produce IgE antibody specific for the injected passive antibody. Immune complexes of IgE bound to the antibody can mediate systemic mast cell degranulation, leading to systemic anaphylaxis. Another possibility is that the recipient will produce IgG or IgM antibodies specific for the foreign antibody, which will form complement-activating immune complexes. The deposition of these complexes in the tissues can lead to type III hypersensitive reactions.
In addition, the small polypeptides in the venom are frequently not able to elicit a strong immunogenic reaction from the host. Potent neurotoxins, which often are relatively small and low abundance molecules, may not always induce the production of sufficient quality and quantity of antibody molecules. Therefore, a balance between the injected dose, the toxicity towards the subject animal and high quality antibody production has to be obtained, often empirically, every time a new batch of antivenom is produced. Identification of less abundant, but highly potent components in a purified venom mixture has advantages, compared to using the crude venom as antigen to raise antibodies for therapeutic purposes.
Scorpion venoms contain many small protein neurotoxins that act selectively on various types of voltage-gated ion channels. These neurotoxins affect the victim by interfering with neuronal ionic balance and channel activity. Ion channels are multi-subunit, membrane bound proteins critical for maintenance of cellular homeostasis in nearly all cell types. Channels are involved in a myriad of processes including modulation of action potentials, regulation of cardiac myocyte excitability, heart rate, vascular tone, neuronal signaling, activation and proliferation of T-cells, and insulin secretion from pancreatic islet cells. In humans, ion channels comprise extended gene families with hundreds, or perhaps thousands, of both closely related and highly divergent family members. The majority of known channels regulate the passage of sodium (Na+), chloride (Cl−), calcium (Ca++) and potassium (K+) ions across the cellular membrane.
Binding of scorpion toxins to target ion channels is known to occur through multiple interactions (Rogers et al. (1996)J. Biol. Chem. 271:15950–15962) Numerous amino acid residues have been determined to have effect on binding (Possani et al. (1999) Eur. J. Biochem. 264:287–300). In addition, alpha scorpion toxins are known to slow or inhibit sodium channel inactivation. Recently their mechanism of action at the molecular level on sodium channels became more apparent. These site 3 binding toxins bind to the extracellular S3-S4 loop of the domain IV, a major part of the voltage sensor, on the sodium channel and alter the transmembrane movement of this region which is required in the gating process (Cestele and Catterall (2000) Biochimie (Paris) 82:883–892.).
Given their importance in maintaining normal cellular physiology, it is not surprising that ion channels have been shown to play a role in heritable human disease. Indeed, ion channel defects are involved in predisposition to epilepsy, cardiac arrhythmia (long QT syndrome), hypertension (Bartter's syndrome), cystic fibrosis, (defects in the CFTR chloride channel), several skeletal muscle disorders (hyperkalemic periodic paralysis, paramyotonia congenita, episodic ataxia) and congenital neural deafness (Jervell-Lange-Nielson syndrome), among others.
Recently, a toxin called margatoxin was isolated from the venom of Centruroides margaritatus. Margatoxin is very potent and selectively binds to one subtype of potassium channel produced by human T-lymphocytes (Lin et al. (1993) J. Exp. Med. 177:637–645). Margatoxin may be useful in treating autoimmune diseases or in preventing the rejection of organ transplants (WO 95/03065). Another neurotoxin known in the art is Botox®, or botulinum toxin type A, which is a muscle-relaxing agent that works at the motor nerve endings. Botox® is used in treating neuromuscular problems, cervical dystonia, strabismus and blepharospasm. Botox® is also used in the cosmetic dermatology industry to prevent wrinkle formation (see U.S. Pat. No. 5,721,215). Although these toxins and many others have been useful as experimental tools, they are not particularly selective in their actions on different tissues and they affect a variety of subtypes of ion channels.
In addition to their effects on ion channels, scorpion venoms are also known to modulate the kinin pathway in animals. Kinins are nonapeptides generated as a result of the activity of killikreins, a group of proteolytic enzymes present in most tissues and body fluids, on kinonogens. Once released, kinins such as bradykinin and related peptides kallikin (Lys-bradykinin) and Met-Lys-bradykinin produce many physiological responses, including pain and hyperanalgesia, in addition to contributing to the inflammatory response (reviewed in Couture et al Eur. J. Pharm. 429:161–176 2001 and Campbell et al Clin. Exp. Pharm. Phys. 28: 1060–1065 2001). In addition, bradykinin is overproduced in a very wide range of pathological conditions, and is thought to be a contributing factor in septic shock, asthma, and can also increase the permeability of the blood-brain barrier and thereby promote the passage of anti-infectious or antitumoral drugs. How scorpions modulate the kinin pathway in animals is, as yet, unknown.
Accordingly, there is a need to characterize the compositions of scorpion toxins not only in order to develop more effective antivenoms, but also to understand human and animal physiological responses to the venoms. The characterization of particular toxins that are involved in ion channel regulation or kinin responses are of particular interest as ion channels and kinins are involved in many other conditions and diseases. The present invention addresses these needs and many others.
Also of interest are the following publications: WO 00/78958, EP 1185654, WO 00/78957, EP 1185653, WO 00/32777, WO 00/24772, EP1124954, Couture et al, European J. Pharmacology 429 161–176, 2001; Kotovych et al Biochem. Cell Bio. 76:257–266, 1998; Campbell, Clinical and Experimental Pharmacology and Physiology 28:1060–1065, 2001; and Ferreira et al., Toxicon 36:31–39, 1998.